U.S. patent number 10,081,662 [Application Number 15/304,434] was granted by the patent office on 2018-09-25 for modified relaxin b chain peptides.
This patent grant is currently assigned to THE FLOREY INSTITUTE OF NEUROSCIENCE AND MENTAL HEALTH. The grantee listed for this patent is The Florey Institute of Neuroscience and Mental Health. Invention is credited to Ross Alexander David Bathgate, Mohammed Akhter Hossain, John Desmond Wade.
United States Patent |
10,081,662 |
Bathgate , et al. |
September 25, 2018 |
Modified relaxin B chain peptides
Abstract
Provided herein are biologically active single chain relaxin
peptides. In particular the present invention relates to single
chain relaxin peptides comprising a B chain derived from relaxin-2,
the peptide being truncated by one or more amino acid residues at
the N-terminus with respect to the sequence of the B chain of
native relaxin-2. Typically the single chain relaxin peptides
selectively bind to the RXFP1 receptor.
Inventors: |
Bathgate; Ross Alexander David
(Monee Ponds, AU), Hossain; Mohammed Akhter
(Brunsiwck West, AU), Wade; John Desmond (Canterbury,
AU) |
Applicant: |
Name |
City |
State |
Country |
Type |
The Florey Institute of Neuroscience and Mental Health |
Parkville |
N/A |
AU |
|
|
Assignee: |
THE FLOREY INSTITUTE OF
NEUROSCIENCE AND MENTAL HEALTH (Parkville, Victoria,
AU)
|
Family
ID: |
54323290 |
Appl.
No.: |
15/304,434 |
Filed: |
April 17, 2015 |
PCT
Filed: |
April 17, 2015 |
PCT No.: |
PCT/AU2015/050184 |
371(c)(1),(2),(4) Date: |
October 14, 2016 |
PCT
Pub. No.: |
WO2015/157829 |
PCT
Pub. Date: |
October 22, 2015 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20170037106 A1 |
Feb 9, 2017 |
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Foreign Application Priority Data
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Apr 17, 2014 [AU] |
|
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2014901409 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K
14/64 (20130101); A61P 9/00 (20180101); A61K
38/00 (20130101) |
Current International
Class: |
C07K
14/00 (20060101); C07K 14/46 (20060101); C07K
14/435 (20060101); C07K 14/47 (20060101); C12N
15/16 (20060101); C12N 15/12 (20060101); C12N
15/11 (20060101); C12N 15/09 (20060101); C12N
15/00 (20060101); C07K 14/64 (20060101); C07K
14/575 (20060101); A61K 38/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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102643825 |
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Aug 2012 |
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CN |
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WO-2009055854 |
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May 2009 |
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WO |
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WO-2013007563 |
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Jan 2013 |
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WO |
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Other References
Tokuriki et al., 2009, Curr. Opin. Struc. Biol. 19:596-604). cited
by examiner .
Bhattacharya et al. (2017, PLoS ONE 12(3): e0171355,
https://doi.org/10.1371/journal.pone.0171355). cited by examiner
.
International Search Report issued PCT/AU2015/050184 on May 27,
2015. cited by applicant .
Eigenbrot, et al,. X-ray Structure of Human Relaxin at 1 5 a
Comparison to Insulin and Implications for Receptor Binding
Determinants, J. Mol. Biol. 1991, 221:15-21. cited by applicant
.
Haugaard-Kedstrom, et al., Design, Synthesis, and Characterization
of a Single-Chain Peptide Antagonist for the Relaxin-3 Receptor
RXFP3, J. Am. Chem. Soc., 2011, 133:4965-4974. cited by applicant
.
Hossain, et al., The Minimal Active Structure of Human Relaxin-2,
The Journal of Biological Chemistry, 2011, 286:43:37555-37565.
cited by applicant .
Hossain, et al., The Roles of the A- and B-Chains of Human
Relaxin-2 and -3 on Their Biological Activity, Current Protein and
Peptide Science, 2010, 11(8):719-724. cited by applicant .
Silvertown, et al., Analog of H2 Relaxin Exhibits Antagonistic
Properties and Impairs Prostate Tumor Growth, The FASEB Journal,
2006, 21(3):754-765. cited by applicant .
Supplementary European Search Report issued in EP15780000 on Aug.
31, 2017. cited by applicant.
|
Primary Examiner: Kemmerer; Elizabeth
Attorney, Agent or Firm: Blank Rome LLP
Claims
The invention claimed is:
1. A single chain relaxin peptide having relaxin-2 activity
comprising a relaxin B chain derived from relaxin-2, wherein the
peptide is truncated by six amino acid residues at the N-terminus
with respect to the sequence of the B chain of native relaxin-2
shown in SEQ ID NO: 1, wherein the peptide comprises four
additional amino acids at the C-terminus, at least two of which are
positively charged, and wherein one or both cysteine residues at
positions 11 and 23, with respect to the native human relaxin-2 B
chain sequence of SEQ ID NO: 1, is replaced with a neutral amino
acid.
2. A peptide according to claim 1, wherein the additional amino
acids at the C-terminus comprise KRSL (amino acid residues 30-32 of
SEQ ID NO: 3).
3. A peptide according to claim 1, wherein one or both cysteine
residues at positions 11 and 23 is replaced with serine or
alanine.
4. A single chain relaxin peptide having relaxin-2 activity
comprising a relaxin B chain derived from relaxin-2, wherein the
peptide is truncated by six amino acid residues at the N-terminus
with respect to the sequence of the B chain of native relaxin-2
shown in SEQ ID NO: 1. wherein the peptide comprises four
additional amino acids at the C-terminus, at least two of which are
positively charged, and wherein one or both cysteine residues at
positions 11 and 23, with respect to the native human relaxin-2 B
chain sequence of SEQ ID NO: 1, is replaced with a neutral amino
acid, wherein one or more arginine residues of the native human
relaxin-2 B chain sequence are replaced by a basic amino acid.
5. A peptide according to claim 4, wherein the basic amino acid is
lysine, or a mimetic or isostere of arginine.
6. A peptide according to claim 5, wherein said mimetic or isostere
is homoarginine, norarginine or guanidine propionic acid.
7. A peptide according to claim 4, wherein the arginine residue at
position 17 of the native human relaxin-2 B chain sequence shown in
SEQ ID NO: 1 is replaced by lysine, homoraginine, norarginine or
guanidine propionic acid.
8. A peptide according to claim 1, wherein the peptide comprises or
consists of the amino acid sequence shown in any one of SEQ ID NO:
7, 9, 10 or 11.
9. A peptide according to claim 1, wherein the peptide comprises a
C-terminal amide group and/or an N-terminal acetyl group.
10. A peptide according to claim 1, wherein the peptide is
selective for the RXFP1 receptor.
11. A peptide according to claim 1, wherein the peptide is an
agonist of the RXFP1 receptor.
12. A polynucleotide encoding a single chain relaxin peptide
according to claim 1.
13. A pharmaceutical composition comprising a single chain relaxin
peptide according to claim 1, optionally together with one or more
pharmaceutically acceptable carriers, excipients or diluents.
14. A composition comprising a polynucleotide of claim 12,
optionally together with one or more pharmaceutically acceptable
carriers, excipients or diluents.
Description
This application is the U.S. national stage of International Patent
Application No. PCT/AU2015/050184, filed Apr. 17, 2015, which
claims the benefit of Australian Patent Application No. 2014901409,
filed Apr. 17, 2014.
FIELD OF THE INVENTION
The present invention relates generally to biologically active
single chain relaxin peptides and to nucleic acids encoding the
same. The present invention in particular relates to single chain
relaxin-2 peptides comprising a relaxin-2 derived B chain and which
optionally selectively bind to the RXFP1 (LGR7) receptor. The
invention also relates to uses of peptides of the invention,
methods employing the same and to compositions comprising such
peptides.
BACKGROUND OF THE INVENTION
Relaxins are heterodimeric peptide hormones composed, in their
mature form, of an A chain and a B chain linked via disulfide
bridges. Human relaxins in their mature form are typically
stabilised by three disulfide bonds, two inter-chain disulfide
bonds between the A chain and B chain and one intra-chain disulfide
bond between cysteine residues in the A chain.
Relaxins have been conserved through vertebrate evolution and have
been characterised in a large and diverse range of vertebrate
species. In particular the cysteine residues in the B and A chains
responsible for the intra- and inter-chain disulfide bonds are
highly conserved. Whilst in most species only two forms of relaxin
have been identified (relaxin and relaxin-3), in humans three
distinct forms of relaxin have been described and the genes and
polypeptides characterised. These have been designated H1, H2 and
H3, Homologues of H1 and H2 relaxin have been identified in other
higher primates including chimpanzees, gorillas and orangutans.
Differing expression patterns for H1, H2 and H3 relaxin suggest
some differences in biological roles, however all three forms
display similar biological activities, as determined for example by
their ability to modulate (stimulate or inhibit) cAMP activity in
cells expressing relaxin family receptors, and accordingly share
some biological functions in common.
The biological actions of relaxins are mediated through G protein
coupled receptors. To date, H1, H2 and H3 relaxins have been shown
to primarily recognise and bind four receptors, RXFP1 (LGR7), RXFP2
(LGR8), RXFP3 (GPCR135) and RXFP4 (GPCR142). Receptors RXFP1 and
RXFP2 are structurally distinct from receptors RXFP3 and RXFP4, yet
despite the differences there is significant cross-reactivity
between different native relaxin molecules and different
receptors.
Initially thought to be predominantly a reproductive hormone, it
has become increasingly clear that human relaxin-2 has pleiotropic
actions. Relaxin-2 has been shown to have potent cardioprotective
(including vasodilatory and angiogenic) effects and antifibrotic
effects (see, for example, Du et al., 2010, Nat. Rev. Cardiol, 7,
48-58 and Samuel, 2005, Clin. Med. Res. 3, 241-249). Relaxin-2 is
currently undergoing clinical trial evaluation for the treatment of
acute heart failure.
With the increasing therapeutic promise shown by relaxin-2 and the
continued development of potential clinical applications there is
also an interest in developing relaxin peptides that are simpler in
structure than native relaxin molecules and yet which retain the
ability to bind to relaxin receptors and/or retain
relaxin-associated biological activity. Simplifying the structure
of therapeutic peptides and minimising the amino acid sequence
required to impart biological activity on therapeutic peptides can
serve to reduce the cost of polypeptide synthesis, reduce the
complexity and difficulty of synthesis, and/or improve the
efficiency of synthesis. Moreover, simplified, smaller molecules
may exhibit improved in vivo activities and/or cellular uptake of
such molecules may be improved when compared to native
counterparts. In addition, improvements to pharmacokinetic
properties (such as half-life, bioavailability etc) and/or
therapeutic efficacy may be more readily made to simplified,
smaller peptides.
SUMMARY OF THE INVENTION
Provided herein are novel, modified relaxin peptides that comprise
only relaxin-2-derived B chain and which retain biological activity
associated with native relaxin-2. Peptides of the invention are
"modified" in that they possess B chain amino acid sequences that
differ from those found in corresponding native relaxin-2 molecules
at one or more positions.
A first aspect of the invention provides a biologically active
single chain relaxin peptide comprising a relaxin B chain derived
from relaxin-2, wherein the peptide is truncated by one or more
amino acid residues at the N-terminus with respect to the sequence
of the B chain of native relaxin-2.
Typically the native relaxin-2 comprises or consists of the
sequence shown in SEQ ID NO: 1.
The peptide may be truncated by, for example, up to about seven
residues at the N-terminus. The peptide may comprise or consist of
the amino acid sequence shown in SEQ ID NO:5, or a variant or
derivative thereof.
The peptide may comprise one or more additional amino acids at the
C-terminus. The one or more additional amino acids may increase the
solubility of the peptide when compared to the native relaxin-2 B
chain. One or more of the additional amino acids may be positively
charged amino acids. The peptide may comprise four additional amino
acids at the C-terminus. The additional amino acids may be KRSL.
The peptide may comprise or consist of the amino acid sequence
shown in SEQ ID NO:6, or a variant or derivative thereof.
One or more cysteine residues in the native relaxin-2 sequence may
be replaced with a neutral amino acid, for example serine or
alanine, more typically serine. For example, the cysteine residues
at positions 11 and 23 of the native human relaxin-2 B chain
sequence shown in SEQ ID NO:1 may be replaced by serine residues.
The peptide may comprise or consist of the amino acid sequence
shown in SEQ ID NO:7, or a variant or derivative thereof, or SEQ ID
NO:8, or a variant or derivative thereof.
One or more arginine residues of the native human relaxin-2 B chain
sequence may be replaced by a basic amino acid. The basic ammo acid
may be lysine, or a mimetic or isostere of arginine. Said mimetic
or isostere may, for example, be homoarginine, norarginine or
guanidine propionic acid. The arginine residue at position 17 of
the native human relaxin-2 B chain sequence shown in SEQ ID NO:1
may be replaced by lysine, homoraginine, norarginine or guanidine
propionic acid. The peptide may comprise or consist of the amino
acid sequence shown in SEQ ID NO:9, or a variant or derivative
thereof, SEQ ID NO:10, or a variant or derivative thereof, or SEQ
ID NO:11, or a variant or derivative thereof.
The single chain peptide typically comprises a C-terminal amide or
acid group, more typically a C-terminal amide group. The single
chain peptide may comprise an N-terminal acetyl group.
The peptide may he selective or specific for the RXFP1 receptor.
The peptide may be an agonist of the RXFP1 receptor. The peptide
may be a selective or specific agonist of the RXFP1 receptor.
A second aspect of the invention provides a polynucleotide encoding
a modified biologically active single chain relaxin peptide
according to the first aspect.
A third aspect of the invention provides a pharmaceutical
composition comprising a biologically active single chain relaxin
peptide of the first aspect, or a polynucleotide of the second
aspect, optionally together with one or more pharmaceutically
acceptable carriers, excipients or diluents.
A fourth aspect provides a method for treating or preventing a
disease or condition, the method comprising administering to a
subject in need thereof a biologically active single chain relaxin
peptide of the first aspect, a polynucleotide of the second aspect
or a pharmaceutical composition of the third aspect.
The disease or condition may be fibrosis or a cardiovascular
disease or condition. The fibrosis may be renal fibrosis, cardiac
fibrosis or pulmonary fibrosis. The cardiovascular disease or
condition may be acute heart failure, coronary artery disease,
cardiac fibrosis or microvascular disease.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention is described, by way of non-limiting example
only, with reference to the accompanying drawings.
FIG. 1. Solubility of H2 relaxin B chains (at concentration 4
mg/ml). The native H2 B chain is insoluble (left vial), whereas the
B7-33 C11.23S peptide is soluble (right vial).
FIG. 2. Binding of native H2 relaxin and single B chain B7-33
C11.23S peptide (B7-33) to RXFP1-expressing cells (A) and to 7BP
cells (B) in the presence of europium (Eu)-labelled H2 relaxin.
FIG. 3. (A) cAMP-stimulating activity of single chain B7-33 C11.23S
peptide (B7-33; inverted triangles) compared to native H2 relaxin
(H2 relaxin; squares) in RXFP1-expressing cells. (B)
cAMP-stimulating activity of single chain B7-33 C11.23S peptide
(B7-33; inverted triangles) compared to native H2 relaxin (H2
relaxin; squares) and the native ligand of RXFP2 INSL3 (diamonds)
in RXFP2-expressing cells.
FIG. 4. Time course of ERK1/2 activation by H2 relaxin (H2 relaxin,
diamonds) or B7-33 C11.23S (B7-33, circles) in rat renal
myofibroblasts.
FIG. 5. Single chain B7-33 C11.23S peptide (B7-33) promotes
expression of MMP-2 to similar levels as native H2 relaxin in both
(A) rat renal myofibroblasts and (B) human cardiac fibroblasts, as
demonstrated by gelatin zymography (a, c) and densitometry (b, d).
Data shown (b and d) are mean.+-.SEM (n=3-5 separate experiments).
***p<0.001 vs untreated control.
FIG. 6. Effect of H2 relaxin and B7-33 C11.23S peptide (B7-33)
treatment on cardiac fibrosis and function in a rat model of
myocardial infarction. Picrosirius red stained left ventricles of
the heart from vehicle (Veh). H2 relaxin (1-12) and B7-33 C11.23S
treated rats (A). H2 relaxin and B7-33 C11.23S significantly reduce
the percentage of collagen in the interstitial area compared with
vehicle treated rats (B). Left ventricular end-diastolic pressure
(LVEDP) is reduced in animals treated with H2 relaxin and B7-33
C11.23S compared to vehicle-treated animals 12 weeks after
myocardial infarction (C). **p<0.01 vs control group; *p<0.05
vs control group.
FIG. 7. Effect of H2 relaxin and B7-33 C11.23S peptide (B7-33)
treatment on cardiac fibrosis in a mouse model of isoproterenol
(ISO) induced cardiac fibrosis. Picrosirius red stained left
ventricles of the heart from vehicle, ISO, ISO and H2 relaxin, and
ISO and B7-33 C11.23S treated mice (A). Percentage interstitial
collagen content, determined by picrosirius red staining (B), and
total left ventricular collagen concentration, determined by
hydroxyproline analysis (C) demonstrate that ISO significantly
increases left ventricular collagen density and concentration. The
ISO-related increase in collagen is reduced by treatment with H2
relaxin or B7-33 C11.23S. **p<0.01, ***p<0.001 vs control
group, ##p<0.01 vs ISO group.
FIG. 8. Effect of H2 relaxin and B7-33 C11.23S peptide (B7-33)
treatment on measures of fibrosis in a mouse model of OVA-induced
chronic allergic airway disease. Masson trichrome-stained lung
airway sections of vehicle, OVA and vehicle, OVA and H2 relaxin,
and OVA and B7-33 C11.23S treated mice (A), scale bar=300 .mu.m. H2
relaxin and B7-33 C11.23S normalise subepithelial collagen
thickness (.mu.m) in the lamina reticularis after treatment with
OVA (B). Total lung collagen content (a measure of fibrosis),
measured by hydroxyproline ananlysis, is also normalised in the H2
relaxin and B7-33 C11.23S treated groups relative to OVA treatment
(C). Airway resistance measured in saline, OVA and vehicle, OVA and
H2 relaxin and OVA and B7-33 C11.23S groups in response to
increasing concentrations of the bronchoconstrictor, methacholine,
by invasive plethysmography. Error bars represent SEM.
***p<0.001 vs control group, .sup.#p<0.05, .sup.##p<0.01,
.sup.##p<0.001 vs OVA group.
FIG. 9. Tumour development 10 days after injection with 5000RM1
mouse prostate tumour cells into prostates of C57B6J mice (A).
Tumour development was promoted by H2 relaxin, but not B7-33
C11.23S peptide (B7-33) (B). *p<0.05 vs untreated. SV: seminal
vesicle, PT: prostate tumour.
The subject specification contains amino acid sequence information
prepared using the programme PatentIn Version 3.5, presented herein
in a Sequence Listing. Amino acid sequences are referred to by a
sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond
numerically to the sequence identifiers <400>1 (SEQ ID NO:1),
<400>2 (SEQ ID NO:2), etc. Sequences of the various peptides
are listed in Table 1.
DETAILED DESCRIPTION OF THE INVENTION
The articles "a" and "an" are used herein to refer to one or to
more than one (i.e., to at least one) of the grammatical object of
the article. By way of example, "an element" means one element or
more than one element.
Throughout this specification and the claims which follow, unless
the context requires otherwise, the word "comprise", and variations
such as "comprises" or "comprising", will be understood to imply
the inclusion of a stated integer or step or group of integers or
steps but not the exclusion of any other integer or step or group
of integers or steps.
In the context of this specification, the term "about," is
understood to refer to a range of numbers that a person of skill in
the art would consider equivalent to the recited value in the
context of achieving the same function or result.
The term "peptide" means a polymer made up of amino acids linked
together by peptide bonds. The term "polypeptide" may also be used
to refer to such a polymer although in some instances a polypeptide
may be longer i.e. composed of greater amino acid residues) than a
peptide. Nevertheless, the terms "polypeptide" and "peptide" may be
used interchangeably herein.
The term "relaxin peptide" as used herein means a peptide, whether
modified in accordance with the present invention or corresponding
to a naturally occurring relaxin molecule which displays biological
activity typically associated with relaxin. The level of such
relaxin biological activity displayed by a modified peptide of the
invention may be equivalent to that of a naturally occurring or
native relaxin, or may be enhanced or reduced when compared with
the activity of a naturally occurring or native relaxin. In the
context of the present disclosure, the term "single chain relaxin
peptide" refers to peptides comprising only a relaxin B chain
sequence.
The term "modified" as used herein in the context of a relaxin
peptide means a peptide that differs from a naturally occurring or
native relaxin peptide at one or more amino acid positions of such
naturally occurring or native peptide.
The term "conservative amino acid substitution" as used herein
refers to a substitution or replacement of one amino acid for
another amino acid with similar properties within a peptide chain.
For example, the substitution of the charged amino acid glutamic
acid (Glu) for the similarly charged amino acid aspartic acid (Asp)
would be a conservative amino acid substitution. The nature of
other conservative amino acid substitutions is well known to those
skilled in the art.
The term "native" as used herein in relation to relaxin peptides
refers to naturally occurring or wild-type molecules. In various
contexts the terms "native" and "naturally occurring" may refer to
a relaxin peptide as encoded by, and produced from, the genome of
an organism. For example in the context of the present disclosure,
the term "native human relaxin-2" or "native H2 relaxin" refers to
the native or naturally occurring human relaxin-2 molecule, being a
heterodimer comprising an A and a B chain. The amino acid of the B
chain of native H2 relaxin may be as shown in SEQ ID NO:1. The term
"native" also refers to various alternative forms (e.g.
post-translationally modified) in which the naturally occurring or
wild-type molecule may be found, and the term "native" encompasses
such alternative forms.
As used herein the term "derived" in the context of B chains in
modified peptides means that the B chain sequence corresponds to,
originates from, or otherwise shares significant sequence homology
with a naturally occurring relaxin B chain sequence. Those skilled
in the art will understand that by being "derived" from a naturally
occurring or native relaxin sequence, the sequence in the modified
peptide need not be physically constructed or generated from the
naturally occurring or native sequence, but may be chemically
synthesised such that the sequence is "derived" from the naturally
occurring or native sequence in that it shares sequence homology
and function with naturally occurring or native sequence.
As used herein the term "selective" when used in the context of the
ability of a modified relaxin peptide to bind a particular
receptor, for example the RXFP1 (LGR7) receptor, means that the
peptide binds that receptor at significantly higher frequency than
it binds other receptors, for example the RXFP2 receptor. A
modified relaxin peptide that is "specific" for a particular
receptor is one that possesses no discernable activity at any other
receptor. Thus, a modified relaxin polypeptide that is "specific"
for RXFP1 is, by definition, selective for RXFP1.
The term "polynucleotide" as used herein refers to a single- or
double-stranded polymer of deoxyribonucleotide, ribonucleotide
bases or known analogues of natural nucleotides, or mixtures
thereof, that encodes a peptide or polypeptide. The term includes
reference to the specified sequence as well as to the sequence
complimentary thereto, unless otherwise indicated. The terms
"polynucleotide" and "nucleic acid" may be used interchangeably
herein.
As used herein the terms "treating", "treatment", "preventing" and
"prevention" refer to any and all uses which remedy a condition or
symptoms, prevent the establishment of a condition or disease, or
otherwise prevent, hinder, retard, or reverse the progression of a
condition or disease or other undesirable symptoms in any way
whatsoever. Thus the terms "treating" and "preventing" and the like
are to be considered in their broadest context. For example,
treatment does not necessarily imply that a patient is treated
until total recovery. Similarly, "prevention" dose not necessarily
mean that the subject will not eventually contract a particular
condition or disease. Rather, "prevention" encompasses reducing the
severity of, or delaying the onset of, a particular condition or
disease. In the context of some conditions, methods of the present
invention involve "treating" the condition in terms of reducing or
eliminating the occurrence of a highly undesirable and irreversible
outcome of the progression of the condition but may not of itself
prevent the initial occurrence of the condition. Accordingly,
treatment and prevention include amelioration of the symptoms of a
particular condition or preventing or otherwise reducing the risk
of developing a particular condition.
As used herein the terms "effective amount" and "effective dose"
include within their meaning a non-toxic but sufficient amount or
dose of an agent or compound to provide the desired effect. The
exact amount or dose required will vary from subject to subject
depending on factors such as the species being treated, the age and
general condition of the subject, the severity of the condition
being treated, the particular agent being administered and the mode
of administration and so forth. Thus, it is not possible to specify
an exact "effective amount" or "effective dose". However, for any
given case, an appropriate "effective amount" or "effective dose"
may be determined by one of ordinary skill in the art using only
routine experimentation.
Human relaxin-2 (H2 relaxin) is an insulin-like peptide, comprising
two chains (A and B chains) and three disulfide bonds. The A chain
contains 24 residues whereas the B chain may have length variations
(B1-29, B1-31 and B1-33) at the C-terminus. The recombinant H2
relaxin currently under human clinical trials (Serelaxin; RLX030)
for the treatment of acute heart failure contains 29 residues in
the B-chain. In the context of the present specification, this
B1-29 containing H2 relaxin is referred to as "native H2 relaxin"
or "native human relaxin-2", and the B1-29 B chain is referred to
as "native H2 relaxin B chain" or "native human relaxin-2 B chain".
A typical amino acid sequence of the native H2 relaxin B chain is
shown in SEQ ED NO:1.
This native H2 relaxin B chain (SEQ ID NO:1) has an overall net
charge of zero (four positively charged and four negatively charged
amino acids) and is insoluble in aqueous solution, making chemical
synthesis and purification difficult. In contrast, an extended B
chain sequence with an additional four amino acids (KRSL) at the
C-terminus (B1-33) has an overall charge of +2 (six positively
charged and four negatively charged amino acids), thereby imparting
improved solubility compared with the native H2 relaxin B chain.
Once the B chain (either native or B1-33) is chemically combined
with the A chain with three-disulfide connectivity, the resulting
H2 relaxin molecules become very soluble. However the cost and
efficiency, inter alia, of synthesis of a heterodimeric molecule is
a hindrance to the large scale production of native relaxin-2 for
therapeutic purposes and for the formulation of suitable
pharmaceutical compositions.
As described and exemplified herein the present inventors have
synthesised modified, single relaxin B chain peptides that are
soluble and retain biological activity associated with relaxin-2.
In particular embodiments these peptides are shorter than the
native relaxin-2 B chain. Accordingly, being considerably simpler
in structure, the peptides of the present invention offer numerous
advantages for over longer relaxin molecules and over the native
relaxin-2 molecule in terms of production of molecules and
pharmaceutical composition formulation.
Provided herein are modified, single chain relaxin peptides
possessing biological activity associated with relaxin-2, and that
are optionally capable of selectively or specifically binding and
activating the RXFP1 receptor. According to one aspect of the
present invention, there is provided biologically active single
chain relaxin peptides comprising a relaxin B chain derived from
relaxin-2, wherein the peptides are truncated by one or more amino
acid residues at the N-terminus with respect to the sequence of the
B chain of native relaxin-2. In particular, provided herein are
single chain peptides truncated by up to about 7 amino acids at the
N-terminus of the relaxin-2 B chain compared to the native
relaxin-3 B chain sequence, and optionally incorporating up to
about 4 additional amino acids at the C-terminus. Optionally the
peptides also comprise one or more amino acid modifications within
the peptide chain replacing, for example, cysteine residue(s) with
neutral amino acids, and/or replacing arginine residue(s) with
mimetics or isosteres.
The truncation of relaxin peptides, the addition of amino acids and
the replacement of amino acid residues may be achieved in any one
of a number of ways as will be apparent to those skilled in the
art, using approaches and methodologies well known to those skilled
in the art.
The single B chain relaxin peptides of the present invention do not
include a relaxin- or relaxin superfamily member-derived A chain.
However those skilled in the art will appreciate that the term
"single B chain relaxin peptide", and variations thereof, simply
refers to the absence of an A chain. Peptides of the present
disclosure may be combined with or linked to (by covalent or other
means) one or more additional proteinaceous or non-proteinaceous
moieties as may be desirable depending on the use to which the
relaxin peptide of the invention is to be put.
The B chain of native H2 relaxin comprises the amino acid sequence
depicted in SEQ ID NO.1. Accordingly, the B chain amino acid
sequences of single chain relaxin peptides the subject of the
present invention may be based on, or derived from, the amino acid
sequence of the H2 relaxin B chain, for example the sequence
depicted in SEQ ID NO:2. However those skilled in the art will also
appreciate that the amino acid sequences of B chains from which the
modified peptides of the invention may be based, or from which the
modified peptides may be derived, may include variants of this H2
relaxin B chain sequence.
The term "variant" as used herein refers to substantially similar
sequences. Generally, peptide sequence variants also possess
qualitative biological activity in common, such as receptor binding
activity. Further, these peptide sequence variants may share at
least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% sequence identity. Also included
within the meaning of the term "variant" are homologues of peptides
of the invention. A homologue is typically a peptide from a
different species but sharing substantially the same biological
function or activity as the corresponding peptide disclosed herein.
Further, the term "variant" also includes analogues of the peptides
of the present invention, wherein the term "analogue" means a
peptide which is a derivative of a peptide of the invention, which
derivative comprises addition, deletion, substitution of one or
more amino acids, such that the peptide typically retains
substantially the same function, for example in terms of receptor
binding activity. Amino acid insertional derivatives include amino
and/or carboxylic terminal fusions as well as intrasequence
insertions of single or multiple amino acids. Insertional amino
acid sequence variants are those in which one or more amino acid
residues are introduced into a predetermined site in a peptide
although random insertion is also possible with suitable screening
of the resulting product. Deletional variants are characterised by
the removal of one or more amino acids from the sequence.
Substitutional amino acid variants are those in which at least one
residue in a sequence has been removed and a different residue
inserted in its place. Additions to amino acid sequences may
include fusions with other peptides, polypeptides, proteins or
polymers. Modifications may be made to relaxin polynucleotide
sequences, for example via the insertion or deletion of one or more
codons, such that modified derivatives of the relaxin polypeptide
are generated. Such modifications are also included within the
scope of the term "variant". For example, modifications may be made
so as to enhance the biological activity or expression level of the
relaxin or to otherwise increase the effectiveness of the peptide
to achieve a desired outcome.
Single B chain peptides of the invention are modified with respect
to the native H2 relaxin sequence such that the peptide sequence is
truncated by one or more amino acids at the N-terminus. For
example, one, two, three, four, five, six, seven or more amino
acids may be deleted from the N-terminus provided the resulting
peptide retains biological activity in common with native
relaxin-2, for example in terms of RXFP1 receptor binding and
activation activity. Similarly, single B chain peptides of the
invention may be modified with respect to the native H2 relaxin
sequence such that the peptide sequence comprises one or more
additional amino acids at the C-terminus. For example, one, two,
three, four, or more amino acids may be added to the C-terminus
provided the resulting peptide retains biological activity in
common with native relaxin-2, for example in terms of RXFP1
receptor binding and activation activity.
One or more cysteine residues in the native relaxin-2 sequence may
be replaced by neutral amino acids, such as serine or alanine
residues, more typically serine. In a particular embodiment, where
the cysteine residues at positions 11 and 23 (or corresponding
positions) of the native human relaxin-2 sequence are replaced by
serine residues. The single chain polypeptide may further comprise
the replacement of one or more arginine residues in the native
relaxin-2 sequence with one or more basic amino acids. The basic
amino acid may be lysine, or a mimetic or isostere of arginine,
such as, for example homoarginine, norarginine or guanidine
propionic acid. In a particular embodiment the arginine residue at
position 17 of the native human relaxin-2 B chain sequence is
replaced by lysine, homoraginine, norarginine or guanidine
propionic acid.
The single B chain peptides of the invention typically comprises an
amide (for example --NH.sub.2) or acid (for example --OH) group on
the exposed end of the C-terminal amino acid residue. In particular
embodiments this C-terminal group is an amide group, typically
NH.sub.2.
As described and exemplified herein a single B chain relaxin
peptide in accordance with the present invention may comprise or
consist of an amino acid sequence as set forth in any one of SEQ ID
Nos 5 to 11, or a variant or derivative thereof. Those skilled in
the art will however appreciate and recognise that the scope of the
present disclosure is not limited to the specific single B chain
relaxin peptide sequences exemplified herein, but rather other
sequences having the general sequence characteristics set our
herein are also contemplated and encompassed.
Therefore, those skilled in the art will appreciate that amino acid
sequence modifications additional to these specifically exemplified
herein may also be made. Exemplary amino acid changes may include:
the replacement of the isoleucine residue at position 20 of the
native H2 relaxin B chain sequence of SEQ ID NO:1 with a mimetic or
isostere thereof; the replacement of one or more amino acids with
non-native amino acid equivalents such as beta-alanine in place of
alanine); and the replacement of non helix-inducing residues (such
as valine or proline) with helix-inducing native or non-native
amino acids (Ala, Aib etc).
The present inventors have previously demonstrated that mutations
of arginine residues at positions 13 and 17 in the B chain of
relaxin-2 can generate an RXFP1 antagonist peptide (Hossain M A et
at. 2010 Amino Acids 39::409-16; Silvertown et al. 2007 FASEB J.
21:754-65). Accordingly, embodiments of the present invention
provide antagonists of RXFP1 wherein peptides disclosed herein
contain mutations at positions Arg13 or Arg17 of the native human
relaxin-2 B chain sequence shown in SEQ ID NO:1. The arginine
residues may be replaced by, for example, lysine residues or
arginine mimetics. By way of example, the single B chain peptide
B7-33 exemplified herein may be modified by replacing the Arg13
residue with a lysine residue or an arginine mimetic or isostere
such as homoarginine.
Relaxin peptides further modified at the N- and/or C-terminus by
the addition, deletion or substitution of one or more amino acid
residues also fall within the scope of the present invention. Such
modifications may, for example, improve the solubility of the
peptide. For example, the C-terminus may be extended by the
addition of, or two or more C-terminal residues may be replaced
with, two or more charged residues such as KK, RR or KR.
Such amino acid changes may be effected by synthesis of peptide
sequences (such as, but not limited to the method exemplified
herein). Alternatively, recombinant DNA and nucleotide replacement
techniques may be used which include the addition, deletion or
substitution of nucleotides (conservative and/or non-conservative),
under the proviso that the proper reading frame is maintained. A
conservative substitution denotes the replacement of an amino acid
residue by another, biologically similar residue. Examples of
conservative substitutions include the substitution of one
hydrophobic residue such as isoleucine, valine, leucine, alanine,
cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine,
norleucine or methionine for another, or the substitution of one
polar residue for another, such as the substitution of arginine for
lysine, glutamic acid for aspartic acid, or glutamine for
asparagine, and the like. Neutral hydrophilic amino acids which can
be substituted for one another include asparagine, glutamine,
serine and threonine. The term "conservative substitution" also
includes the use of a substituted amino acid in place of an
unsubstituted parent amino acid. Exemplary techniques for
generating such amino acid insertion, deletion or substitution
modifications include random mutagenesis, site-directed
mutagenesis, oligonucleotide-mediated or polynucleotide-mediated
mutagenesis, deletion of selected region(s) through the use of
existing or engineered restriction enzyme sites, and the polymerase
chain reaction. Such techniques will be well known to those skilled
in the art.
Peptides of the invention can also be further modified, for
instance, by glycosylation, amidation, carboxylation, or
phosphorylation, or by the creation of acid addition salts, amides,
esters, in particular C-terminal esters, and N-acyl derivatives.
The peptides can also be further modified to create peptide
derivatives by forming covalent or non-covalent complexes with
other moieties. Covalently-bound complexes can be prepared by
cross-linking the chemical moieties to functional groups on the
side chains of amino acids comprising the peptides, or at the N-or
C terminus. For example, as peptide sequence minimisation may be
accompanied by increased susceptibility to enzymatic attack and
degradation with a corresponding decrease in plasma half life and
in vivo activity, a modified peptide of the present invention may
be generated with a polyethylene moiety conjugated at one or more
locations (PECiylation) to increase in vivo half life of the
peptide. Those skilled in the art will appreciate that a number of
other well known approaches exist to extend the in vivo half life
of peptides, such as for example the addition of albumin affinity
tags, lipidation (fatty acid conjugation), XTENylation, PASylation,
oligomerization and the present disclosure is not limited by
reference to the exemplary means specifically discussed herein.
The structures of the peptides of the invention may be stabilised
through amino acid modifications and subsequent reactions to, for
example, induce intra-peptide bonds which may or may not increase
potency of the peptide. Some embodiments of the invention provide
for alterations of the structure of the peptides including, by way
of example only, by head to tail cyclization through amide bonds
using appropriate spacer and side-chain to side-chain cyclization
and "stapling" through bonds, including but not limited to lactam
bonds, disulfide bonds, thioether bonds, or diselenide bonds.
Methods for generating such structures are well known to those
skilled in the art.
Further, the peptides of the present invention can be conjugated to
a reporter group, including, but not limited to a radiolabel, a
fluorescent label, an enzyme (e.g., that catalyzes a colorimetric
or fluorometric reaction), a substrate, a solid matrix, or a
carrier (e. g., biotin or avidin). These are merely exemplary
additional modifications that may be made to the modified peptides
of the invention. Those skilled in the art will appreciate that
further modifications may also be made so as to generate analogues
of the peptides of the invention. By way of example only,
illustrative analogues and processes for preparing the same are
described in International patent application published as WO
2004/113381, the disclosure of which is incorporated herein by
reference in its entirety.
Amino acid additions may also result from the fusion of a relaxin
peptide or fragment thereof with a second peptide, such as a
polyhistidine tag, maltose binding protein fusion, glutathione S
transferase fusion, green fluorescent protein fusion, or the
addition of an epitope tag such as FLAG or c-myc.
Peptides of the invention may be synthesised by standard methods of
liquid or solid phase chemistry well known to those of ordinary
skill in the art. For example such molecules may be synthesised
following the solid phase chemistry procedures of Steward and Young
(Steward, J. M. & Young, J. D., Solid Phase Peptide Synthesis.
(2nd Edn.) Pierce Chemical Co., Illinois, USA (1984), or Howl (ed.)
Peptide Synthesis and Applications, Methods in Molecular Biology
(Volume 298), 2005. In general, such synthesis methods comprise the
sequential addition of one or more amino acids or suitably
protected amino acids to a growing peptide chain. Typically, either
the amino or carboxyl group of the first amino acid is protected by
a suitable protecting group. The protected amino acid is then
either attached to an inert solid support or utilised in solution
by adding the next amino acid in the sequence having the
complimentary (amino or carboxyl) group suitably protected and
under conditions suitable for forming the amide linkage. The
protecting group is then removed from this newly added amino acid
residue and the next (protected) amino acid is added, and so forth.
After all the desired amino acids have been linked, any remaining
protecting groups, and if necessary any solid support, is removed
sequentially or concurrently to produce the final polypeptide.
Peptides of the invention may also be produced using standard
techniques of recombinant DNA and molecular biology that are well
known to those skilled in the art. Guidance may be obtained, for
example, from standard texts such as Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989 and
Ausubel et al., Current Protocols in Molecular Biology, Greene
Publ. Assoc. and Wiley-Intersciences, 1992, Methods described in
Morton et al., 2000 (Immumnol Cell Biol 78:603-607), Ryan et al.,
1995 (J Biol Chem 270:22037-22043) and Johnson et al., 2005 (J Biol
Chem 280:4037-4047) are examples of suitable purification methods
for relaxin peptides, although the skilled addressee will
appreciate that the present invention is not limited by the method
of purification or production used and any other method may be used
to produce relaxin peptides for use in accordance with the methods
and compositions of the present invention.
Relaxin peptide fragments may be produced by digestion of a
polypeptide with one or more proteinases such as endoLys-C,
endoArg-C, endoGlu-C, and staphylococcus V8-protease. The digested
peptide fragments can be purified by, for example, high performance
liquid chromatographic (HPLC) techniques. The purification of
modified relaxin polypeptides of the present disclosure may be
scaled-up for large-scale production purposes. For this purpose a
range of techniques well known to those skilled in the art are
available.
Embodiments of the present disclosure also provide isolated
polynucleotides encoding relaxin peptides of the invention. Those
skilled in the art will appreciate that heterologous expression of
polypeptides may be improved by optimising the codons for the
particular species in which the relaxin polypeptide is to be
expressed. Accordingly, polynucleotides encoding relaxin peptides
of the invention may be codon-optimised for expression in a
particular species.
In particular embodiments, polynucleotides may be cloned into a
vector. The vector may be a plasmid vector, a viral vector, or any
other suitable vehicle adapted for the insertion of foreign
sequences, their introduction into eukaryotic cells and the
expression of the introduced sequences. Typically the vector is a
eukaryotic expression vector and may include expression control and
processing sequences such as a promoter, an enhancer, ribosome
binding sites, polyadenylation signals and transcription
termination sequences.
The present invention also provides antibodies that selectively
bind to the modified relaxin peptides of the invention, as well as
variants, fragments and analogues thereof. Suitable antibodies
include, but are not limited to polyclonal, monoclonal, chimeric,
humanised, single chain. Fab fragments, and an Fab expression
library. Antibodies of the present invention may act as agonists or
antagonists of relaxin polypeptides, or fragments or analogues
thereof. Methods for the generation of suitable antibodies will be
readily appreciated by those skilled in the art. For example, an
anti-relaxin monoclonal antibody, typically containing Fab
portions, may be prepared using the hybridoma technology described
in Antibodies-A Laboratory Manual, Harlow and Lane, eds., Cold
Spring Harbor Laboratory, N.Y. (1988).
Screening for the desired antibody can also be accomplished by a
variety of techniques known in the art. Assays for immunospecific
binding of antibodies may include, but are not limited to,
radioimmunoassays. ELISAs (enzyme-linked immunosorbent assay),
sandwich immunoassays, immunoradiometric assays, gel diffusion
precipitation reactions, immunodiffusion assays, in situ
immunoassays, Western blots, precipitation reactions, agglutination
assays, complement fixation assays, immunofluorescence assays,
protein A assays, and immunoelectrophoresis assays, and the like
(see, for example, Ausubel et al., eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York).
Antibody binding may be detected by virtue of a detectable label on
the primary anti-relaxin antibody. Alternatively, the anti-relaxin
antibody may be detected by virtue of its binding with a secondary
antibody or reagent which is appropriately labelled. A variety of
methods are known in the art for detecting binding in an
immunoassay and are within the scope of the present invention.
Single B chain relaxin peptides of the present invention find
particular application as in the study of relaxin biological
activities and as therapeutic agents. Polynucleotides encoding the
peptides and antibodies to the peptides find similar application.
Therapeutic applications include, but are not limited to, the
treatment of fibrosis and fibrotic disorders and of cardiovascular
disorders. For example, peptides of the invention may find
application in the treatment of renal fibrosis, pulmonary fibrosis,
cardiac fibrosis, coronary artery disease, acute heart failure,
microvascular disease, preeclampsia, hypertensive diseases,
scleroderma, cervical ripening, fibromyalgia and in orthodontics.
However those skilled in the art will appreciate that the scope of
the present disclosure is not limited to these uses, and peptides
of the invention will find application in the treatment of any
disease, condition or disorder against which native relaxin-2 may
be considered.
In general, suitable compositions for use with the methods of the
invention may be prepared according to methods and procedures that
are known to those of ordinary skill in the art and accordingly may
include pharmaceutically acceptable carriers, diluents and/or
adjuvants.
Compositions may be administered by standard routes. In general,
the compositions may be administered by the parenteral (e.g.,
intravenous, intraspinal, subcutaneous or intramuscular), oral or
topical route. Administration may be systemic, regional or local.
The particular route of administration to be used in any given
circumstance will depend on a number of factors, including the
nature of the condition to be treated, the severity and extent of
the condition, the required dosage of the particular compound to be
delivered and the potential side-effects of the compound.
In general, suitable compositions may be prepared according to
methods which are known to those of ordinary skill in the art and
may include a pharmaceutically acceptable diluent, adjuvant and/or
excipient. The diluents, adjuvants and excipients must be
"acceptable" in terms of being compatible with the other
ingredients of the composition, and not deleterious to the
recipient thereof.
Examples of pharmaceutically acceptable carriers or diluents are
demineralised or distilled water; saline solution; vegetable based
oils such as peanut oil, safflower oil, olive oil, cottonseed oil,
maize oil, sesame oils such as peanut oil, safflower oil, olive
oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut
oil; silicone oils, including polysiloxanes, such as methyl
polysiloxane, phenyl polysilloxane and methylphenyl polysolpoxane;
volatile silicones; mineral oils such as liquid paraffin, soft
paraffin or squalane; cellulose derivatives such as methyl
cellulose, ethyl cellulose, carboxymethylcellulose, sodium
carboxymethylcellulose or hydroxypropylmethylcellulose; lower
alkanols, for example ethanol or iso-propanol; lower aralkanols;
lower polyalkylene glycols or lower alkylene glycols, for example
polyethylene glycol, polypropylene glycol, ethylene glycol,
propylene glycol, 1,3-butylene glycol or glycerin; fatty acid
esters such as isopropyl palmitate, isopropyl myristate or ethyl
oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth or
gum acacia, and petroleum jelly. Typically, the carrier or carriers
will form from 10% to 99.9% by weight of the compositions.
Compositions may be in a form suitable for administration by
injection, in the form of a formulation suitable for oral ingestion
(such as capsules, tablets, caplets, elixirs, for example), in the
form of an ointment, cream or lotion suitable for topical
administration, in a form suitable for delivery as an eye drop, in
an aerosol form suitable for administration by inhalation, such as
by intranasal inhalation or oral inhalation, in a form suitable for
parenteral administration, that is, subcutaneous, intramuscular or
intravenous injection.
For administration as an injectable solution or suspension,
non-toxic parenterally acceptable diluents or carriers can include,
Ringer's solution, isotonic saline, phosphate buffered saline,
ethanol and 1,2 propylene glycol.
Some examples of suitable carriers, diluents, excipients and
adjuvants for oral use include peanut oil, liquid paraffin, sodium
carboxymethylcellulose, methylcellulose, sodium alginate, gum
acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol,
gelatine and lecithin. In addition these oral formulations may
contain suitable flavouring and colourings agents. When used in
capsule form the capsules may be coated with compounds such as
glyceryl monostearate or glyceryl distearate which delay
disintegration.
Adjuvants typically include emollients, emulsifiers, thickening
agents, preservatives, bactericides and buffering agents.
Methods for preparing parenterally administrable compositions are
apparent to those skilled in the art, and are described in more
detail in, for example, Remington's Pharmaceutical Science, 15th
ed., Mack Publishing Company, Easton, Pa., hereby incorporated by
reference herein.
The composition may incorporate any suitable surfactant such as an
anionic, cationic or non-ionic surfactant such as sorbitan esters
or polyoxyethylene derivatives thereof Suspending agents such as
natural gums, cellulose derivatives or inorganic materials such as
silicaceous silicas, and other ingredients such as lanolin, may
also be included.
The compositions may also be administered in the form of liposomes.
Liposomes are generally derived from phospholipids or other lipid
substances, and are formed by mono- or multi-lamellar hydrated
liquid crystals that are dispersed in an aqueous medium. Any
non-toxic, physiologically acceptable and metabolisable lipid
capable of forming liposomes can be used. The compositions in
liposome form may contain stabilisers, preservatives, excipients
and the like. The preferred lipids are the phospholipids and the
phosphatidyl cholines (lecithins), both natural and synthetic.
Methods to form liposomes are known in the art, and in relation to
this specific reference is made to: Prescott, Ed., Methods in Cell
Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33
et seq., the contents of which is incorporated herein by
reference.
For the purposes of the present invention administration may be
therapeutic or preventative. In therapeutic applications,
compositions are administered to a patient already suffering from a
disease, in an amount sufficient to cure or at least partially
arrest the disease and its complications. The composition should
provide a quantity of the molecule or agent sufficient to
effectively treat the patient. The therapeutically effective dose
level for any particular patient will depend upon a variety of
factors including: the disorder being treated and the severity of
the disorder; activity of the molecule or agent employed; the
composition employed; the age, body weight, general health, sex and
diet of the patient; the time of administration; the route of
administration; the rate of sequestration of the molecule or agent;
the duration of the treatment; drugs used in combination or
coincidental with the treatment, together with other related
factors well known in medicine.
One skilled in the art would be able, by routine experimentation,
to determine an effective, non-toxic amount of agent or compound
which would be required to treat applicable diseases and
conditions. Generally, an effective dosage is expected to be in the
range of about 0.0001 mg to about 1000 mg per kg body weight per 24
hours; typically, about 0.001 mg to about 750 mg per kg body weight
per 24 hours; about 0.01 mg to about 500 mg per kg body weight per
24 hours; about 0.1 mg to about 500 mg per kg body weight per 24
hours; about 0.1 mg to about 250 mg per kg body weight per 24
hours; about 1.0 mg to about 250 mg per kg body weight per 24
hours. More typically, an effective dose range is expected to be in
the range about 1.0 mg to about 200 mg per kg body weight per 24
hours; about 1.0 mg to about 100 mg per kg body weight per 24
hours; about 1.0 mg to about 50 mg per kg body weight per 24 hours;
about 1.0 mg to about 25 mg per kg body weight per 24 hours; about
5.0 mg to about 50 mg per kg body weight per 24 hours; about 5.0 mg
to about 20 mg per kg body weight per 24 hours; about 5.0 mg to
about 15 mg per kg body weight per 24 hours.
Alternatively, an effective dosage may be up to about 500 m.sup.2.
Generally, an effective dosage is expected to be in the range of
about 25 to about 500 mg/m.sup.2, preferably about 25 to about 350
mg/m.sup.2, more preferably about 25 to about 300 mg/m.sup.2, still
more preferably about 25 to about 250 mg/m.sup.2, even more
preferably about 50 to about 250 mg/m.sup.2, and still even more
preferably about 75 to about 150 mg/m.sup.2.
Further, it will be apparent to one of ordinary skill in the art
that the optimal quantity and spacing of individual dosages will be
determined by the nature and extent of the disease state being
treated, the form, route and site of administration, and the nature
of the particular individual being treated. Also, such optimum
conditions can be determined by conventional techniques.
It will also be apparent to one of ordinary skill in the art that
the optimal course of treatment, such as, the number of doses of
the composition given per day for a defined number of days, can be
ascertained by those skilled in the art using conventional course
of treatment determination tests.
Those skilled in the art will appreciate that in accordance with
the methods of the present disclosure relaxin peptides may be
administered alone or in conjunction with one or more additional
agents. Additionally, the present disclosure contemplates
combination therapy using relaxin peptides disclosed herein in
conjunction with other therapeutic approaches to the treatment of
diseases and disorders. For such combination therapies, each
component of the combination therapy may be administered at the
same time, or sequentially in any order, or at different times, so
as to provide the desired effect. Alternatively, the components may
be formulated together in a single dosage unit as a combination
product. When administered separately, it may be preferred for the
components to be administered by the same route of administration,
although it is not necessary for this to be so.
Embodiments of the present invention also contemplate the
administration of a polynucleotide encoding a single B chain
relaxin peptide of the invention. In such situations the
polynucleotide is typically operably-linked to a promoter such that
the appropriate peptide sequence is produced following
administration of the polynucleotide to the subject. The
polynucleotide may be administered to subjects in a vector. The
vector may be a plasmid vector, a viral vector, or any other
suitable vehicle adapted for the insertion of foreign sequences,
their introduction into eukaryotic cells and the expression of the
introduced sequences. Typically the vector is a eukaryotic
expression vector and may include expression control and processing
sequences such as a promoter, an enhancer, ribosome binding sites,
polyadenylation signals and transcription termination sequences.
The nucleic acid construct to be administered may comprise naked
DNA or may be in the form of a composition, together with one or
more pharmaceutically acceptable carriers.
The reference in this specification to any prior publication (or
information derived from it), or to any matter which is known, is
not, and should not be taken as an acknowledgment or admission or
any form of suggestion that that prior publication (or information
derived from it) or known matter forms part of the common general
knowledge in the field of endeavour to which this specification
relates.
The present invention will now be described with reference to the
following specific examples, which should not be construed as in
any way limiting the scope of the invention.
EXAMPLES
Example 1
Single Chain Relaxin-2 Peptide Construction
The amino acid sequences of single B chain relaxin-2 molecules
according to the present invention are shown in Table 1. The
sequences are also provided in the formal Sequence Listing
appearing at the end of this specification under the SEQ ID Nos
indicated in the table.
TABLE-US-00001 TABLE 1 Amino acid sequences of relaxin-2 B chains.
Residues in bold replace cysteine residues at positions 11 and 23
in native human relaxin-2 B chain (SEQ ID NO: 1). Amino acids added
to the C- terminus of the native relaxin-2 peptide are underlined.
Residues replacing the arginine residue at position 17 of native
human relaxin-2 B chain (SEQ ID NO: 1) are double underlined. Ac-
indicates N-terminal acetylation of the peptide. SEQ ID NO: Peptide
Sequence 1 H2 B chain DSWMEEVIKLCGRELVRAQIAICGMSTWS-NH.sub.2 2
B1-29 C11.23S DSWMEEVIKLSGRELVRAQIAISGMSTW-NH.sub.2 3 B1-33
DSWMEEVIKLCGRELVRAQIAICGMSTWSKRSL-NH.sub.2 4 B1-33 C11.23S
DSWMEEVIKLSGRELVRAQIAISGMSTWSKRSL-NH.sub.2 5 B7-29
VIKLCGRELVRAQIAICGMSTWS-NH.sub.2 6 Native B7-33
VIKLCGRELVRAQIAICGMSTWSKRSL-NH.sub.2 7 Modified B7-33
VIKLSGRELVRAQIAISGMSTWSKRSL-NH.sub.2 (B7-33 C11.23S) 8 B7-29
C11.23S VIKLSGRELVRAQIAISGMSTWS-NH.sub.2 9 B7-33 C11.23S
VIKLSGRELVKAQIAISGMSTWSKRSL-NH.sub.2 R17K 10 B7-33 C11.23S
VIKLSGRELVhRAQIAISGMSTWSKRSL-NH.sub.2 R17HR 11 B7-33 C11.23S
VIKLSGRELVnRAQIAISGMSTWSKRSL-NH.sub.2 R17NR 12 AcB7-33 C11.23S
Ac-VIKLSGRELVRAQIAISGMSTWSKRSL-NH.sub.2 13 R13A (AcB7-33
Ac-VIKLSGAELVRAQIAISGMSTWSKRSL-NH.sub.2 C11.23S) 14 R17A (AcB7-33
Ac-VIKLSGRELVAAQIAISGMSTWSKRSL-NH.sub.2 C11.23S) 15 I20A (AcB7-33
Ac-VIKLSGRELVRAQAAISGMSTWSKRSL-NH.sub.2 C11.23S) 16 R13/17A
Ac-VIKLSGAELVAAQAAISGMSTWSKRSL-NH.sub.2 I20A (AcB7-33 C11.23S) 17
KKKK (AcB7-29 Ac-VIKLSGRELVRAQIAISGMSTWSKKKK-NH.sub.2 C11.23S) 1 hR
= homoarginine 2 nR = norarginine
Solid-Phase Peptide Synthesis:
Synthetic single chain relaxin-2 B chain peptides were generated by
solid phase peptide synthesis. The synthesis of derivatives of
human relaxin-2 B chain was achieved using Fmoc-methodology as
previously described (Dawson et al. J Peptide Res 53:542-547, 1999)
with or without using microwave energy. The solid support was
Fmoc-PAL PEG-PS (PerSeptive Biosystems, USA), and HBTU-activated
Fmoc-amino acids were used throughout. Fmoc deprotection was with
20% piperidine in DMF. All derivatives were purchased from Auspep
(Melbourne, Australia). Cleavage of the peptides from the solid
support and side chain deprotection was achieved by a 2 hour
treatment with trifluoroacetic acid (ITA) in the presence of
phenol, thioanisole, ethanedithiol and water (82.5/5/5/2.5/5, v/v).
The crude peptides were subjected to reversed-phase high
performance liquid chromatography (RP-HPLC) on a Vydac C18 column
(Hesperia, USA) using a 1%/min gradient of CH3CN in 0.1% aqueous
TEA for analysis. Some polypeptides were oxidised in a buffer
containing 1 mM DPDS for 1 hour and the reaction monitored on HPLC
and by mass spectrometry.
Peptide Characterization
Polypeptides were purified using RP-HPLC systems using a
preparative column while the final purity of individual synthetic
peptides was assessed by analytical RP-HPLC using a Vydac C18
column (250.times.4.6 mm, 300 .ANG., 5 .mu.m) with a buffer system
of 0.1% trifluoroacetic acid in water (buffer A) and 0.1%
trifluoroacetic acid in acetonitrile (buffer B). The molecular
weights of all analogues were determined by MAIDI-TOF mass
spectrometry using a Bruker AutoflexII instrument in the linear
mode at 19.5 kV. Furthermore, the peptide content for each analogue
was quantitated by amino acid analysis using vapour-phase acid
hydrolysis in 6 M hydrochloric acid containing 2% phenol at
110.degree. C. over 24 hours. The hydrolysate was then converted to
stable, fluorescent derivatives using a Waters AccQTag kit. The
derivitized amino acids were separated using a Shim-Pak XR ODS
column (3.times.75 mm, 2.2 .mu.m) on a Shimadzu microbore RP-HPLC
system.
The inventors have previously shown that truncation of six residues
from the N-terminus of the native relaxin-2 B chain (SEQ ID NO:1)
does not affect RXFP1 activity indicating that these residues are
not functionally important. Truncation of six residues (including
three negatively charged amino acids and three hydrophobic
residues) from the N-terminus of the native relaxin-2 B chain
provides the resulting B chain peptide, (B7-29; SEQ ID NO:5), with
an overall positive charge (+3) and improved aqueous solubility.
Truncation of six residues from the N-terminus of the native
relaxin-2 B chain together with addition of four residues (two
positively charged) at the C-terminus yields the peptide, B7-33
(SEQ ID NO:6), with an overall positive charge (+5), and fewer
hydrophobic residues. This highly charged peptide is freely
water-soluble (FIG. 1).
Example 2
Ligand Binding Activities and cAMP Response Stimulation
Human embryonic kidney (HEK-293T) cells stably transfected with
RXFP1 were cultured in RPMI 1640 medium supplemented with 10% fetal
calf serum, 100 .mu.g/ml penicillin, 100 .mu.g/ml streptomycin and
2 mM L-glutamine and plated into 96-well pre-coated with
poly-L-lysine for whole cell binding assays. Competition binding
experiments were conducted with Eu.sup.3+-labelled H2 relaxin (as
per Shabanpoor et al., 2012, Biochem Biophys Res Commun. 420,
253-256) in the absence or presence of increasing concentrations of
unlabelled relaxin-2 B chain derivatives. Nonspecific binding was
determined with an excess of unlabelled peptides (500 nM H2
relaxin). Fluorescent measurements were recorded at an excitation
wavelength of 340 nm and emission of 614 nm on a Victor plate
reader (Perkin-Elmer Inc.). FIG. 2A demonstrates that B7-33 C11.23S
binds RXFP1, but with lower affinity than H2 relaxin. Binding of
native B7-33, B7-33 C11.23S and N-terminal acetylated B chain
peptide derivatives to cells expressing a fusion protein comprising
the extracellular domain of RXFP1 and the transmembrane domain of
CD8 (7BP cells) demonstrates that the B chain peptide derivatives
strongly bind to 7BP cells, however still with a lower affinity
than H2 relaxin (Table 2, FIG. 2B). Statistical differences in
pIC.sub.50 values were analyzed using one-way analysis of variance
coupled to Newman Keul's multiple comparison test for multiple
group comparisons in GraphPad Prism 6.
The ability of the relaxin-2 B chain peptide derivatives generated
in Example 1 to stimulate cAMP response was also evaluated, using a
cAMP reporter gene assay as described previously (Scott et al.,
2006, J Biol Chem. 281, 34942-34954). HEK-293T cells co-transfected
with either RXFP1 or RXFP2, or 7BP cells, and a pCRE
.beta.-galactosidase reporter plasmid were plated in 96-well
plates. After 24 hours, the co-transfected cells were incubated
with increasing concentrations of peptides in parallel to 10 nM of
H2 relaxin or INSL3 for RXFP1- or RXFP2-transfected cells
respectively. The amount of cAMP-driven .beta.-galactosidase
expression in each well was assessed with a colorimetric assay
measuring absorbance at 570 nm on a microplate spectrophotometer.
Ligand-induced cAMP stimulation was expressed as a percentage of
maximal response of H2 relaxin or INSL3 for RXFP1 and RXFP2 cells
respectively. Each data point was measured in triplicate and each
experiment conducted independently at least three separate times.
Statistical differences in pEC.sub.50 values were analyzed using
one-way ANOVA coupled to Newman Keul's multiple comparison test for
multiple group comparisons in GraphPad Prism 6. Results for cells
transfected with RXFP1 and 7BP cells are shown in Table 2. The
single chain B7-33 C11.23S peptide was also shown to be selective
at RXFP1 over RXFP2 (FIG. 3).
TABLE-US-00002 TABLE 2 Ligand binding of relaxin-2 B chain peptide
derivatives and stimulation of cAMP response by relaxin-2 B chain
peptide derivatives. Values in log M.sup.1. RXFP1 7BP Eu-H2 pKi
cAMP pEC50 Eu-H2 pKi Ligand (n .gtoreq. 3) (n .gtoreq. 3) (n
.gtoreq. 3) H2 relaxin 8.96 .+-. 0.03 10.49 .+-. 0.09 8.97 .+-.
0.10 (6) B1-29 C11.23S <5 <5 -- B1-33ox 6.61 .+-. 0.24# <5
-- B1-33 C11.23S 5.33 .+-. 0.15# 5.10 .+-. 0.06# -- B7-33ox 6.15
.+-. 0.15# 5.11 .+-. 0.11# -- B7-33 C11.23S 5.54 .+-. 0.13# 5.12
.+-. 0.06# 7.65 .+-. 0.10# B7-29 C11.23S <5 <5 -- B7-33
C11.23S R17K <5 <5 -- B7-33 C11.23S 6.52 .+-. 0.07# <5 --
R17HR B7-33 C11.23S <5 5.81 .+-. 0.15# -- R17NR AcB7-33 C11.23S
6.00 (n = 1) 5.40 .+-. 0.04# 7.54 .+-. 0.10# R13A (AcB7-33 <5
<5 5.76 .+-. 0.19# C11.23S) R17A (AcB7-33 <5 <5 6.01 .+-.
0.12# C11.23S) R20A (AcB7-33 <5 <5 -- C11.23S) R13/17A. I20A
<5 <5 5.54 .+-. 0.18# (AcB7-33 C11.23S) KKKK (AcB7-29 6.25
.+-. 0.01# 5.81 .+-. 0.11# 8.91 .+-. 0.08 C11.23S) #p < 0.001 vs
H2 relaxin .sup.1Data are presented as the mean .+-. S.E of the
percentage of the total specific binding of triplicate wells,
repeated in at least three separate experiments, and curves were
fitted using one-site binding curves in GraphPad Prism 6 (GraphPad
Inc, San Diego, CA).
Example 3
Stimulation of Signaling Pathways by B7-33 C11.23S
The inventors then tested the single chain B7-33 C11.23S relaxin-2
peptide for its ability to signal via different pathways, cAMP (see
Example 2) and pERK.1/2 on HEK-293T (cells stably expressing RXFP1)
and myofibroblast cells (cells endogenously expressing RXFP1) The
cAMP assay was conducted as described in Example 2. Phosphorylation
of ERK1/2 was determined using AlphaScreen SureFire.RTM. assay
which is a proprietary, non-radioactive and non-Western proximity
assay that relies on singlet oxygen energy transfer (PerkinElmer
Inc.). Rat renal fibroblasts (that endogenously express RXFP1
receptor) were seeded into a 96-well plate at a density of 40,000
cells per well and incubated overnight in complete media to allow
cell adhesion. Cells were then serum-starved for 4-6 hours followed
by native H2 relaxin (100 nM) or B7-33 C11.23S peptide (100 nM)
treatment for periods of up to 20 minutes and ERK1/2 activation was
quantified using the phospho-ERK1/2 Surefire AlphaScreen kit. B7-33
C11.23S stimulated ERK1/2 with slightly higher level of efficacy as
H2 relaxin peaking at 5 minutes following the peptide treatment
(FIG. 4).
The inventors tested B7-33 in both stably-(HEK-293T) and
natively-expressing (rat renal myofibroblast) RXFP1 cells. At first
the peptide was tested in HEK-RXFP1 cells for its ability to
activate cAMP (FIG. 3A) and EKR signalling pathways (data not
shown) and was found to act as a full agonist, but with poor
potency. Despite the poor potency in HEK-RXFP1 cells, when tested
in rat renal myofibroblast it exhibited very high pERK potency
(FIG. 4).
Example 4
In vitro Anti-fibrotic Activity of B7-33 C11.23S
The inventors then investigated the ability of the single chain
B7-33 C11.23S relaxin-2 peptide to induce matrix
metalloproteinase-2 (VIMP-2) activity, which provides a measure of
the anti-fibrotic activity of the polypeptide.
Renal myofibroblast cells natively expressing human RXFP1 were
plated out onto 12-well plates with a density of 50 000 cells per
well. Cells were treated with 16.8 nM H2-relaxin and 16.80 nM B7-33
C11.23S. Expression of basal MMP-2 levels was monitored with no
treatment applied to wells (control). Each treatment was carried
out in duplicates (n=5), for each replicate in Dulbecco's modified
Eagle's medium (DMEM) containing 10% fetal calf serum, 2.2% HEPES
buffer, 1% L-glutamine and 2% penicillin/strepticillin. The cells
were incubated over 48 hrs in a humidified chamber with 5% CO.sub.2
at 37.8.degree. C. temperature. Following this, media was aspirated
off and experiment continued in serum free media (DMEM containing
2.2% HEPES buffer, 1% L-glutamine, 2% penicillin/strepicillin and
2% lactalbumin hydrolysate) for a further 24 hours in a humidified
chamber with 5% CO.sub.2 at 37.8.degree. C. room temperature. Media
were then collected from various treatments and gel zymography was
carried out.
For determination of MMP-2 expression within various treatments,
zymographic assays of gelatinases were carried out. Polyacrylamide
separating gel containing 7.5% acrylamide, 0.35M Tris-Cl pH8.8,
0.4% SDS, 0.5 mg/ml gelatine solution (porcine skin, 300 bloom),
was stacked below a polyacrylamide stacking gel containing 3.75%
acrylamide, 0.25M Tris-Cl pH6.8, 0.4% SDS. Media from various
treatments were incubated with gel loading sample buffer (0.0625M
Tris-HCl, pH6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue) in
1:4 dilution at room temperature for 1 hr. 25 .mu.L of various
samples were applied to gel lanes. Gels are run at 200V constant
voltage till the dye front nears the end of the gel. The completed
gels were separated from the glass plates and washed twice (15 min
per wash) in 0.25% Triton X-100 (to remove SDS from the gel
proteins). The gels were incubated overnight (>16 hours) with
incubation buffer at 37.degree. C. (0.05M Tris-HCl pH 7.4, 0.01M
CaCl.sub.2, 5% Triton X-100, 0.02% sodium azide, 1 .mu.M
ZnCl.sub.2. The gels were then stained with 0.1% Cootnassie blue
containing 40% 2-propanol and destained with 7% acetic acid before
analysed by ImageJ software.
As shown in FIG. 5, the single chain B7-33 C11.23S relaxin-2
peptide was shown to induce the expression of MMP-2 in rat renal
myofibroblasts and human cardiac fibroblasts to levels similar to
those induced by the native H2 relaxin. It is therefore concluded
that the B7-33 C11.23S relaxin-2 peptide has similar anti-fibrotic
properties as native H2 relaxin.
Example 5
In Vivo Anti-fibrotic Activity of B7-33 C11.23S
To determine the effect of the single chain B7-33 C11.23S relaxin-2
peptide on fibrosis in vivo, the inventors used a rat model of
myocardial infarction, a mouse model of isoproterenol-induced heart
failure, and a mouse model of chronic allergic airways disease
(AAD).
Rats
Adult male Sprague-Dawley rats weighing 250-320 g were obtained
from Animal Resources Centre, Perth, Wash., Australia. The rats
were group housed in a constant temperature of 22.+-.1.degree. C.
and a relative humidity of 50-60% under a controlled light-dark
cycle of 12 hours. Rats were given access to standard laboratory
rat chow and drinking water ad libitum.
All procedures were approved by the Florey Institute Animal Ethics
Committee and were performed in accordance with the Prevention of
Cruelty to Animals Act, Australia 1986 and conformed with
guidelines set out by the National Health and Medical Research
Council of Australia (2007).
Mice
Seven to eight-week old male 129SV mice (which are sensitive to
tissue injury and fibrosis) and age-matched female Balb/c mice
(which are sensitive to changes in airway hyperresponsiveness) were
used for the induction of isoproterenol-induced cardiomyopathy and
ovalbumin-induced chronic allergic airways disease, respectively.
16 five week-old male C57BL/6 mice were used in the induction of
prostate cancer growth. All animals were obtained from Monash
Animal Services (Monash University, Clayton, Victoria, Australia)
and housed under standard conditions (maintained on an 12 h
light-12 h dark lighting cycle with free access to food and water)
in the Department of Pharmacology Animal Room at Monash
University.
All animals were given at least 5 days to acclimatize before any
experimentation was conducted on them. All procedures were approved
by Monash University's Animal Ethics Committees, which adhere to
the Australian Code of Conduct for care and use of laboratory
animals for scientific purposes.
Measurement of Airway Hyperresponsiveness
Twenty-four hours after the last vehicle/drug administration,
methacholine-induced airway reactivity was assessed by invasive
plethysmography as described before (Locke et al. 2007, Am J Respir
Mol Biol 36, 625-632; Royce et al. 2009, Endocrinology 150,
2692-2699). Mice were anaesthetized intraperitoneally with 200
.mu.g/g ketamine and 10 .mu.g/g xylazine. Tracheotomy was performed
using an 18-gauge tracheotomy tube and jugular vein cannulated with
a 0.61 9 0.28 mm polyethylene tube (Microtube Extrusions, North
Rocks, NSW, Australia), Mice were then placed in a plethysmograph
chamber (Buxco Research Systems, Wilmington, N.C., USA) where
increasing concentrations of acetyl-.beta.-methacholine (from 31.25
.mu.g/kg to 500 .mu.g/kg) were delivered intravenously in five
doses. After every dose, airway resistance and compliance were
measured (Biosystem XA version 2.7.9; Buxco Electronics Inc,
Wilmington, N.C., USA). The change in airway resistance calculated
by the maximal resistance after each dose minus baseline resistance
(phosphate buffered saline alone) was plotted against each dose of
methacholine evaluated.
Histopathology
The mid zone of the male mouse heart and largest lung lobe from
female Balb/c mice were fixed in 10% neutral buffered formalin for
24-48 h before being processed and embedded routinely in paraffin
wax. Representative sections of tissue, 3-5 um each, were taken and
stained with either picrosirius red (Samuel et al. 2011 Lab Invest
91, 675-690) for the detection of interstitial collagen or Masson's
trichrome (Royce et al, 2009, Endocrinology 150, 2692-2699) for the
detection of subepithelial basement membrane collagen
deposition).
Morphometric Analysis of Structural Changes
Changes in picrosirius-red stained interstitial collagen or
epithelial thickness and subepithelial collagen (fibrosis) around
the airway lumen from Masson's trichrome-stained sections; which
were all captured (at .times.20 magnification) using a SPOT digital
camera (Q Imaging, Burnaby, BC, Canada) and analysed with Image J
1.3 software (National Institutes of Health, Bethesda, Md.). Four
to five fields per mid zone of the heart or 4-5 airways (of 150-350
.mu.m in diameter) per mouse were assessed. Epithelial thickness
and subepithelial collagen regions were traced with a digital pen
and the thickness of each region calculated by the imaging
software. Results were expressed as mean thickness (1 .mu.m) of the
4-5 airways sampled.
Hydroxyproline Analysis
The apical region of the heart or second largest lung lobe from
each mouse was treated as described previously (Samuel C S, et at.,
2003, FASEB J 17, 121-123; Royce et at. 2009, Endocrinology 150,
2692-2699) for the determination of hydroxyproline content.
Hydroxyproline values were estimated based on a standard curve
constructed with serial dilutions of a 0.1 mg/mL stock of
trans-4-hydroxyproline-L-proline (Sigma-Aldrich). Hydroxyproline
values were then converted to collagen content as detailed
previously (Samuel CS, et al. 2004. Endocrinology 145, 4125-4133)
and, in turn, divided by the dry weight of each corresponding left
ventricular or lung tissue assessed to yield collagen concentration
(a measure of fibrosis).
Statistical Analysis
All data were expressed as the mean+/-SEM and analysed using
GraphPad Prism 6 (GraphPad Software Inc., San Diego, Calif., USA).
The results were analysed by one-way ANOVA, using the Newman-Keuls
post hoc test for multiple comparisons between treatment groups in
all experiments performed except for the analysis of the lung
function (AHR) data, which was assessed by a two-way ANOVA with
Bonferroni's post hoc test. P<0.05 was considered to be
statistically significant.
Myocardial Infarction-induced Heart Failure
Heart failure was induced as previously described (Ruchaya et al.,
2014, Exp Physiol 99, 111-122). Breifly, to induce heart failure,
rats were anaesthetised with an intramuscular injection of ketamine
(60 mg/kg) and medetomide hydrochloride (250 mg/kg). A left sided
thoracotomy through an opening between the fourth and fifth rib was
performed, the heart was exteriorised and the left anterior
descending coronary artery was ligated. Anaesthesia was reversed
with antipamezole hydrocholide (1 mg/kg). Penicillin (1000 U) and
buprenorphine (0.05 mg/kg) was administered to aid post-operative
recovery. Animals were left to recover from the surgery under a
heating source. Rats were individually housed after the
surgery.
Eight weeks after myocardial infarction surgery, rats were randomly
assigned to 3 groups (vehicle, H2 and B7-33), re-anaesthetised
(2-3% isoflurane) and an osmotic mini-pump (model 2ML4, Alzet,
Cupertino, Calif.) implanted intraperitoneally. Vehicle (saline),
native H2 relaxin (0.5 mg/kg/day) or B7-33 C11.23S (0.5 mg/kg/day)
was continuously administered for 28 days. At the conclusion of
treatment, rats were anaesthetized (sodium pentobarbitone, 60 mg/kg
i.p.) and the left ventricular end-diastolic pressure determined
prior to decapitation and removal of the heart for histological
analysis.
Rats treated with either H2 relaxin or B7-33 C11.23S demonstrated a
significant reduction in percentage of collagen in the interstitial
area of the left ventricle as compared with vehicle-treated animals
(FIG. 6A, B). Left ventricular end-diastolic pressure (LVEDP) was
also reduced in H2 relaxin or B7-33 C11.23S treated animals (FIG.
6C), indicating that both H2 relaxin and B7-33 C11.23S treatment
promote a similar improvement in heart function 12 weeks following
myocardial infarction.
Isoproterenol-induced Cardiomyopathy
Male 129SV mice were subcutaneously injected with isoprenaline
hydrochloride (25 mg/kg; Sigma-Aldrich) once daily for 5
consecutive days and then left for a further 9 days for fibrosis
progression to occur. Subgroups of animals (n=7-8/group) received
no treatment (injury alone control) or recombinant H2 relaxin (0.5
mg/mg/day; a dose that had been used previously to successful
demonstrate its anti-fibrotic actions (Samuel C S, et at. 2004,
Endocrinology 145, 4125-4133; Samuel et al. 2011 Lab Invest 91,
675-690) and produce circulating levels of 20-40 ng/ml (Samuel CS,
et al., 2003, FASEB J 17, 121-123), which are well within those
found in pregnant rodents). Alternatively, an equivalent dose of
the B7-33 peptide (0.25 mg/mg/day corrected for MW) was
administered via subcutaneously implanted osmotic minipumps model
2002; Alzet, Cupertino, Calif.), which allowed for the continuous
infusion of each peptide into the circulation of treated animals. A
separate subgroup of mice (n=7) that were not subjected to
isoproterenol or peptide treatment were used as untreated controls.
Nine days after the fifth isoproterenol injection/14 days from the
beginning of the study, all mice were weighed and then sacrificed
for heart and left ventricular collection. A similar portion of the
left ventricle from each animal was then used for the determination
of interstitial collagen staining and morphometric analysis of
interstitial collagen density (Samuel et al. 2011 Lab Invest 91,
675-690) or hydroxyproline content (Samuel C S, et al. 2004,
Endocrinology 145, 4125-4133).
At 14 days, the isoproterenol-treated group displayed a significant
elevation in both percentage of interstitial collagen (FIG. 7A, B)
and total collagen concentration (FIG. 7C) in the left ventricle.
Treatment with H2 relaxin or B7-33 C11.23S significantly reduced
the collagen percentage and total concentration to a similar extent
relative to isoproterenol treatment (FIGS. 7B and 7C) suggesting
that B7-33 C11.23S has similar anti-fibrotic properties to H2
relaxin.
Induction of Chronic Allergic Airway Disease
The inventors also tested the fibrosis preventing activity of B7-33
C11.23S in a model of chronic allergic airway disease (AAD). A
chronic model of ovalbumin (OVA)-induced AAD (Temelkovski et al.
1998, Thorax 53, 849-856) was established in female Balb/c mice
(n=40). Mice were sensitized i.p. on day 0 and 14 with 10 .mu.g
Grade V chicken egg ovalbumin (Sigma-Aldrich Corp., St. Louis, Mo.,
USA) and 0.4 mg aluminium potassium sulphate (alum) in 0.5 mL
saline, then challenged by whole body inhalation exposure to
aerosolized 2.5% OVA (weight/volume of saline) three times a week
from days 21-63 (30 min per session) using an ultrasonic nebulizer
(Locke et al. 2007, Am J Respir Cell Mol Biol 36, 625-632). Control
mice (n=14) were sensitised with 0.4 mg albumin 0.5 mL saline and
challenged with nebulised saline.
Mean airway epithelial thickness was significantly increased by OVA
treatment relative to vehicle treated animals (FIGS. 8A and 8B).
Epithelial thickness was significantly reduced in animals treated
with H2 relaxin or B7-33 C11.23S as compared to OVA treatment, with
H2 relaxin or B7-33 C11.23S administration reducing epithelial
thickness to levels similar to that of vehicle treatment (FIG. 8B).
Similarly, total lung collagen concentration was elevated in OVA
treated animals relative to vehicle treated controls and peptide
treatment following OVA treatment reduced lung collagen relative to
OVA alone (FIG. 8C). Thus the inventors demonstrate that H2 relaxin
and B7-33 C11.23S have similar efficiencies in reducing structural
changes associated with fibrosis. Functionally, OVA significantly
elevates airway hyper-responsiveness as measured by changes in
airway resistance and this increase is attenuated with H2 relaxin
or B7-33 C11.23S treatment (FIG. 8D).
Thus B7-33 C11.23S has similar, albeit slightly diminished
biological activity to that of H2 relaxin in the prevention of
fibrosis and improvement of function in the heart and lung
following chronic and acute disorders of these systems.
Example 6
B7-33 C11.23S Does Not Promote Prostate Tumour Growth
H2 relaxin can induce prostate and other tumour growth. To measure
the effect of B7-33 C11.23S on tumour growth, 16 five week-old male
C57BL/6 mice (obtained from Monash Animal Services) were injected
with 5000 RM1 (mouse prostate tumor) cells into their prostates to
induce tumor growth. One sub-group of mice n=5) was left untreated
until day 10 post-RM1 cell administration. Additional sub-groups of
mice were subcutaneously implanted with osmotic mini-pumps (model
1007D, Durect Corp., Cupertino, Calif., USA) containing H2 relaxin
alone (0.15 mg/kg/day; n=5) or B7-33 C11.23S (0.075 mg/kg/day;
corrected for MW; n=6) on day 2 post-RM1 cell administration and
maintained until day 10 post-cell administration. Each pump had a
reservoir that allowed it to continuously infuse the peptides
administered to mice for 8 days.
H2 relaxin significantly increased tumour size compared to tumours
of untreated mice (FIG. 9). B7-33 C11.23S treatment did not result
in changes in tumour size relative to untreated, and tumours from
B7-33 C11.23S treated mice were significantly smaller than those
from H2 relaxin treated mice (FIG. 9) suggesting that B7-33 C11.23S
may be a safer option than H2 relaxin for therapeutic
administration.
SEQUENCE LISTINGS
1
17129PRTArtificial SequenceSynthetic sequence 1Asp Ser Trp Met Glu
Glu Val Ile Lys Leu Cys Gly Arg Glu Leu Val 1 5 10 15 Arg Ala Gln
Ile Ala Ile Cys Gly Met Ser Thr Trp Ser 20 25 229PRTArtificial
SequenceSynthetic sequence 2Asp Ser Trp Met Glu Glu Val Ile Lys Leu
Ser Gly Arg Glu Leu Val 1 5 10 15 Arg Ala Gln Ile Ala Ile Ser Gly
Met Ser Thr Trp Ser 20 25 333PRTArtificial SequenceSynthetic
sequence 3Asp Ser Trp Met Glu Glu Val Ile Lys Leu Cys Gly Arg Glu
Leu Val 1 5 10 15 Arg Ala Gln Ile Ala Ile Cys Gly Met Ser Thr Trp
Ser Lys Arg Ser 20 25 30 Leu 433PRTArtificial SequenceSynthetic
sequence 4Asp Ser Trp Met Glu Glu Val Ile Lys Leu Ser Gly Arg Glu
Leu Val 1 5 10 15 Arg Ala Gln Ile Ala Ile Ser Gly Met Ser Thr Trp
Ser Lys Arg Ser 20 25 30 Leu 523PRTArtificial SequenceSynthetic
sequence 5Val Ile Lys Leu Cys Gly Arg Glu Leu Val Arg Ala Gln Ile
Ala Ile 1 5 10 15 Cys Gly Met Ser Thr Trp Ser 20 627PRTArtificial
SequenceSynthetic sequence 6Val Ile Lys Leu Cys Gly Arg Glu Leu Val
Arg Ala Gln Ile Ala Ile 1 5 10 15 Cys Gly Met Ser Thr Trp Ser Lys
Arg Ser Leu 20 25 727PRTArtificial SequenceSynthetic sequence 7Val
Ile Lys Leu Ser Gly Arg Glu Leu Val Arg Ala Gln Ile Ala Ile 1 5 10
15 Ser Gly Met Ser Thr Trp Ser Lys Arg Ser Leu 20 25
823PRTArtificial SequenceSynthetic sequence 8Val Ile Lys Leu Ser
Gly Arg Glu Leu Val Arg Ala Gln Ile Ala Ile 1 5 10 15 Ser Gly Met
Ser Thr Trp Ser 20 927PRTArtificial SequenceSynthetic sequence 9Val
Ile Lys Leu Ser Gly Arg Glu Leu Val Lys Ala Gln Ile Ala Ile 1 5 10
15 Ser Gly Met Ser Thr Trp Ser Lys Arg Ser Leu 20 25
1027PRTArtificial SequenceSynthetic
sequenceMOD_RES(11)..(11)homoarginine 10Val Ile Lys Leu Ser Gly Arg
Glu Leu Val Arg Ala Gln Ile Ala Ile 1 5 10 15 Ser Gly Met Ser Thr
Trp Ser Lys Arg Ser Leu 20 25 1127PRTArtificial SequenceSynthetic
sequenceMOD_RES(11)..(11)norarginine 11Val Ile Lys Leu Ser Gly Arg
Glu Leu Val Arg Ala Gln Ile Ala Ile 1 5 10 15 Ser Gly Met Ser Thr
Trp Ser Lys Arg Ser Leu 20 25 1227PRTArtificial SequenceSynthetic
sequenceMOD_RES(1)..(1)ACETYLATION 12Val Ile Lys Leu Ser Gly Arg
Glu Leu Val Arg Ala Gln Ile Ala Ile 1 5 10 15 Ser Gly Met Ser Thr
Trp Ser Lys Arg Ser Leu 20 25 1327PRTArtificial SequenceSynthetic
sequenceMOD_RES(1)..(1)ACETYLATION 13Val Ile Lys Leu Ser Gly Ala
Glu Leu Val Arg Ala Gln Ile Ala Ile 1 5 10 15 Ser Gly Met Ser Thr
Trp Ser Lys Arg Ser Leu 20 25 1427PRTArtificial SequenceSynthetic
sequenceMOD_RES(1)..(1)ACETYLATION 14Val Ile Lys Leu Ser Gly Arg
Glu Leu Val Ala Ala Gln Ile Ala Ile 1 5 10 15 Ser Gly Met Ser Thr
Trp Ser Lys Arg Ser Leu 20 25 1527PRTArtificial SequenceSynthetic
sequenceMOD_RES(1)..(1)ACETYLATION 15Val Ile Lys Leu Ser Gly Arg
Glu Leu Val Arg Ala Gln Ala Ala Ile 1 5 10 15 Ser Gly Met Ser Thr
Trp Ser Lys Arg Ser Leu 20 25 1627PRTArtificial SequenceSynthetic
sequenceMOD_RES(1)..(1)ACETYLATION 16Val Ile Lys Leu Ser Gly Ala
Glu Leu Val Ala Ala Gln Ala Ala Ile 1 5 10 15 Ser Gly Met Ser Thr
Trp Ser Lys Arg Ser Leu 20 25 1727PRTArtificial SequenceSynthetic
sequenceMOD_RES(1)..(1)ACETYLATION 17Val Ile Lys Leu Ser Gly Arg
Glu Leu Val Arg Ala Gln Ile Ala Ile 1 5 10 15 Ser Gly Met Ser Thr
Trp Ser Lys Lys Lys Lys 20 25
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References